Apparatus for the pH determination of blood and method therefor

ABSTRACT

The present invention relates to a spectroscopic apparatus for non-invasive in vivo analysis of blood and to a corresponding analysis method, in particular for direct determination of the pH value of the blood. From a Raman signal detected from an excited target region containing blood at least one predetermined pH sensitive signal portion is determined, from which the pH value of the blood in the target region by use of a relationship between pH value and one or more band parameters of pH sensitive vibrations of at least one molecule or part of a molecule present in blood, in particular heme vibrations of hemoglobin, is determined.

The present invention relates to a spectroscopic analysis apparatus fornon-invasive in vivo analysis of blood and to a corresponding analysismethod, in particular for the determination of the pH value of theblood.

In general, analysis apparatuses, such as spectroscopic analysisapparatuses, are used to investigate the composition of an object to beexamined, e.g. to measure the concentration of various analytes in bloodin vivo. In particular, analysis apparatuses employ an analysis, such asa spectroscopic decomposition, based on interaction of the matter of theobject with incident electromagnetic radiation, such as visible light,infrared or ultraviolet radiation.

A spectroscopic analysis apparatus comprising an excitation system and amonitoring system is known from U.S. Pat. No. 6,609,015, which isincorporated herein by reference. The excitation system emits anexcitation beam to excite a target region during an excitation period.The excitation period and the monitoring period substantially overlap.Hence the target region is imaged together with the excitation, and animage is formed displaying both the target region and the excitationarea. On the basis of this image, the excitation beam can be veryaccurately aimed at the target region.

The analysis method known from U.S. Pat. No. 6,609,015for simultaneousimaging and spectral analysis of a local composition is done by separatelasers for confocal video imaging and Raman excitation or by use of asingle laser for combined imaging and Raman analysis. Orthogonalpolarized spectral imaging (OPS IMAGING), which is also described inU.S. Pat. No. 6,609,015is a simple, inexpensive and robust method tovisualize blood vessels close to the surface of organs which can also beused to visualize blood capillaries in the human skin.

The normal pH range in the blood of humans is 7.25-7.45. Outside thisrange, a person is sick. In critical care units in hospitals doctors usethe pH to look at the acid base status of a patient and oxygenation inrelation to its ventilation. Normally these parameters are measured inso-called blood gas measurements. A quick and exact determination of thepH value is thus of interest. All known methods to determine the pHvalue of blood are, however, indirect measurements, i.e. they are basedon the relationship between pH, bicarbonate and pCO₂. Measuring two ofthese parameters enables the calculation of the third parameter.

U.S. Pat. No. 5,355,880 describes a reliable noninvasive measurement ofblood gases. However, this method is based on absorption differences at2 or more wavelengths. Furthermore the method measures not only inblood.

It is therefore an object of the present invention to provide ananalysis apparatus and a corresponding analysis method for non-invasivein vivo analysis of a patient's blood enabling a direct, quick and exactdetermination of the pH value of the patient's blood, i.e. without theneed to determine bicarbonate or pCO₂.

This object is achieved according to the present invention by ananalysis apparatus comprising:

-   -   an excitation system for emitting an excitation beam to excite a        target region containing blood,    -   a detection system for detecting scattered radiation from the        target region generated by the excitation beam to obtain a Raman        signal,    -   a signal processing system for extracting at least one        predetermined pH sensitive signal portion from said Raman signal        and for determining the pH value of the blood in the target        region from said at least one pH sensitive signal portion by use        of a relationship between pH value and one or more band        parameters of pH sensitive vibrations of at least one molecule        or part of a molecule present in blood.

The present invention is based on the idea to exploit a relationshipbetween pH value and a band parameter of vibrations of a blood molecule.To exploit this relationship a Raman signal of in vivo blood is measurednon-invasively. From this Raman signal at least one pH sensitive signalportion is selected for which the given relationship can be used. Thus,pH sensitive spectral characteristics of the blood sample are determinedfrom which the pH value can be directly derived.

According to the present invention a direct non-invasive measurement ofthe pH value in blood enables a continuous and immediate monitoring incritical care situations, which are useful for e.g. patients withrespiratory or metabolic disorders in the ICU where the condition of thepatient can change rapidly. Further, this method can be applied toneonates who only have a low amount of blood. The replacement ofnon-direct methods may also reduce costs, and finally a reduced risk ofinfections can be achieved.

According to a preferred embodiment of the invention the signalprocessing means is adapted for determining the pH value by use of agiven relationship between pH value and band position, band intensity,band polarisation ratio, band width and/or band shape. This means thatat best only one of these parameters needs to be determined from the pHsensitive signal portion of the detected Raman signal. Since atdifferent wavelengths of the Raman signal relationships between pH valueand different band parameters exist it also depends on the intensity ofthe obtained Raman signal, the magnitude of its pH sensitivity, as wellas on the interfering signal from other molecules in the measurementvolume, which band parameter and which relationship shall be exploitedaccording to the invention in order to obtain the pH value of the blood.pH sensitivity can furthermore be optimized by proper choice of theexcitation wavelength. In that way it is possible to enhance pHsensitive hemoglobin bands over other signal contributions.

According to a preferred embodiment a relationship between pH value andone or more band parameters of heme vibrations of hemoglobin isexploited in order to determine the pH of the blood since the pHsensitivity of the hemoglobin Raman signal is largest. But there areother molecules with pH sensitivity in blood, which can be exploited aswell. It is, for instance, known that molecules that can form H-bridgescan be pH sensitive.

Preferably only one or more Raman signal portions of heme vibrations ofhemoglobin are detected at all instead of detecting a broadband Ramansignal and thereafter extracting predetermined pH sensitive signalportions therefrom. This leads to a simpler, less expensive instrument,comprising just a few optical measurement channels.

It is further preferred to detect only scattered radiation from a targetregion containing essentially whole blood or red blood cells whichcontain hemoglobin which best enables the application of the givenrelationship between pH value and band parameter. However, it isgenerally also possible to perform a measurement on blood plasma orblood serum.

According to a further embodiment a monitoring system for imaging thetarget region and a focussing system for focussing the excitationsystem, the detection system and the monitoring system on the targetregion, in particular on a blood vessel, are provided. By use of thefocussing system the excitation beam can be exactly aimed at the objectof interest, i.e. a blood vessel. Manual or automatic focussingtechniques can be used. To enable a continuous focussing during thewhole analysis, which also compensates for movements of the patientautomatic focussing techniques are preferred.

It is further advantageous if the signal processing system is adaptedfor checking if the at least one pH sensitive signal portion includesinterferences from other analytes, such as lactate and lactate acidosisor oxygenation, and for removing such interferences. This can beachieved by selection of the wavenumber regions used in the analysis orby mathematical modelling of the pH sensitive signal portion and thesignal portion that is sensitive for the analytes, e.g. via spectraprocessing (e.g. filtering and multivariate spectral analysis). Removingthe interferences improves the determination accuracy of the pH valuewith less error and will yield higher pH sensitivity.

According to another aspect of the invention the excitation system isoptimised in the sense of emitting an excitation beam at a wavelengthwhich is optimised such that the at least one pH sensitive signalportion shows an optimum pH sensitivity. Such an optimised excitationwavelength can be found by collecting blood spectra of different pH atdifferent wavelengths and analysing the pH sensitive signal portion. Theoptimum value for the excitation wavelength is characterised by yieldingminimal errors in prediction of the pH in an independent test set ofblood spectra.

Preferred band positions at which the Raman signal shows pH sensitivityare defined in claim 9. At those band positions the relationship betweenpH value and the wavenumber has been found which allows an exactdetermination of the pH value if the wavenumber is determined from theRaman signal. Of course, these band positions are not necessarily theonly band positions, other band positions may be found as well.

The pH influence on the Raman spectra is also revealed in thepolarization characteristics of the vibrational bands of the heme group.The depolarisation ratio, i.e. the ratio of the Raman intensity withperpendicular analyser setting to the intensity with parallel analysersetting with respect to the incoming polarisation orientation, has beenmeasured as a function of the excitation wavelength. These so-calleddepolarisation ratio excitation profiles clearly show pH sensitivity fordifferent band positions, in particular for the same band positions asdefined in claim 9. Since measuring a number of excitation wavelengthsis not very practical, the depolarisation ratio is preferably measuredat a well chosen (one or more) excitation wavelength(s). An appropriateembodiment of the analysis apparatus according to the invention isdefined in claim 10, which makes use of the depolarisation ratio fordetermining the pH value in blood.

The pH dependency of the Raman spectra of blood can also be determinedby application of multivariate statistical calibration techniques, suchas Partial Least Squares (PLS). PLS uses a spectral model data set ofselected blood samples with known pH values, determined with a referenceanalysis method. This model data set should include all possiblespectral variation (both due to pH and interfering contributions ofother analytes) that can be encountered in practice to yield a validmodel. Multivariate analysis of the covariance between the spectra andthe reference values is used to find the spectral regression vector thatcorrelates with the reference pH values. Projection of a new spectrumonto the regression vector then yields the predicted pH value for thatnew spectrum. It is advantageous to use non-linear PLS techniques. Alsoartificial neural networks can be designed and trained by means of modeldata to predict the pH of a new blood sample.

The invention will now be explained in more detail with reference to thedrawings in which

Brief Description of the Drawing

FIG. 1 shows a graphic representation of a preferred embodiment of ananalysis system according to the present invention and

FIG. 2 shows the relationship between pH value and wavenumber at aparticular band position.

FIG. 1 is a graphic representation of a preferred embodiment of ananalysis system in accordance with the invention. The analysis systemincludes an optical monitoring system (lso) for forming an optical imageof the object (obj) to be examined. In the present example the object(obj) is a piece of skin of the forearm of the patient to be examined.The analysis system also includes a multi-photon, non-linear or elasticor inelastic scattering optical detection system (ods) for spectroscopicanalysis of light generated in the object (obj) by a multi-photon ornon-linear optical process. The example shown in FIG. 1 utilises inparticular an inelastic Raman scattering detection system (dsy) in theform of a Raman spectroscopy device. The term optical encompasses notonly visible light, but also ultraviolet radiation and infrared,especially near-infrared radiation.

The monitoring system (lso) comprises a monitoring beam source (ls) foremitting a monitoring beam (irb) and an imaging system (img) for imagingthe target region, e.g. a blood vessel (V) in the upper dermis (D) ofthe patient's forearm (obj). The monitoring beam source (ls) in thisexample comprises a white light source (las), a lense (l1) and aninterference filter (not shown) to produce light in the wavelengthregion of 560-570 nm. Further, a polarizer (p) for polarizing themonitoring beam (irb) is provided. The monitoring beam source (ls) isthus adapted for orthogonal polarized spectral imaging (OPS imaging).

In OPS imaging polarized light is projected by a microscope objective(mo) through a polarizing beam splitter (pbs) onto the skin (obj). Partof the light reflects directly from the surface (specular reflection).Another part penetrates into the skin where it scatters one or moretimes before it is absorbed or is re-emitted from the skin surface(diffuse reflection). In any of these scattering events the polarizationof the incident light is slightly changed. Light that is directlyreflected or penetrates only slightly into the skin will scatter onlyone or a few times before it is re-emitted, and will mostly retain itsinitial polarization. On the other hand, light that penetrates moredeeply into the skin undergoes multiple scattering events and iscompletely depolarized before re-emitted back towards the surface.

When looking at the object (obj) through a second polarizer or analyser(a), oriented precisely orthogonal to that of the first polarizer (p),light reflected from the surface or the upper parts of the skin islargely suppressed, whereas light that has penetrated deep into the skinis mostly detected. As a result the image looks as if it wereback-illuminated. Because wavelengths below 590 nm are strongly absorbedby blood, the blood vessels appear dark in the OPS image.

Generally, an image is obtained using a monochrome CCD camera. Bloodvessels are separated from other absorbing structures be means of size,shape and movement of blood cells. The imaging system (img) used in thepresent embodiment comprises an analyser (a) mentioned above forallowing only light having a polarization orthogonal to the light of thepolarized monitoring beam (irb) to pass which is reflected back throughthe polarizing beam splitter (pbs) from the object (obj). Said light isfurther focused by a lens (l2) onto the CCD-camera (CCD).

The Raman spectroscopy device (ods) comprises an excitation system (exs)for emitting an excitation beam (exb) and a detection system (dsy) fordetection of Raman scattered signals from the target region. Theexcitation system (exs) can be constructed as a diode laser, whichproduces the excitation beam in the form of an 785 nm infrared beam(exb). Of course other lasers can be used as excitation system as well.A system of mirrors and, for instance, a fibre conduct the excitationbeam (exb) to a dichroic mirror (f1) for conducting the excitation beam(exb) along the monitoring beam (irb) to the microscope objective (mo)for focusing both beams onto the object (obj).

The dichroic mirror (f1) also separates the return (monitoring) beamfrom scattered Raman signals. While the reflected monitoring beam istransmitted to the imaging system (img), elastically scattered light andinelastically scattered (Raman) light from the object is reflected atthe dichroic mirror (f1) and conducted back along the light path of theexcitation beam. Inelastically scattered Raman light is then reflectedby an appropriate filter (f2) and directed along the Raman detectionpath in the detection system (dsy) to the input of a spectrometer with aCCD detector. The spectrometer with the CCD detector is incorporatedinto the detector system (dsy), which records the Raman spectrum forwavelengths that are smaller than approximately 1050 nm. It should benotet that this limitation is at present due to limited quantumefficiency at this IR spectral side in CCD cameras. With technicalimprovements in CCD cameras this number 1050 nm can probably beincreased to higher wavelengths.

The output signal of the spectrometer with the CCD detector representsthe Raman spectrum of the Raman scattered infrared light. In practicethis Raman spectrum occurs in the wavelength range beyond 800 nm,depending on the excitation wavelength. The signal output of the CCDdetector is connected to a spectrum display unit, for example aworkstation that displays the recorded Raman spectrum on a monitor. Alsoa calculation unit (e.g. a workstation) is provided to analyse the Ramanspectrum and calculate the concentration of one or more analytes.

Regarding further details of the analysis apparatus in general and thefunction thereof reference is made to the above mentioned WO 02/057759A1.

To achieve continuous auto-focusing of the confocal Raman system (ods)in a blood vessel (V) auto-focussing means (afm) are provided. Suchauto-focussing is required since patients can move during a bloodanalysis in lateral (z) as well as in transversal (x, y) directions.Therefore, continuous determination and adjustment of the optimallocation of the confocal detection centre is required. Transversalmovements can be easily detected by the imaging system, whereas axialmovements are much more difficult to detect. These auto-focussing means(afm) ensure that the optical detection system (ods) and the monitoringsystem (lso) are continuously and optimally focussed on the object ofinterest, e.g. a selected blood vessel (V), during recording of theblood spectra. Many different techniques can be used therefore whichwill not be explained here in detail. Alternatively, manualauto-focussing means can be used instead, by which a user can manuallychange the focussing of the microscope objective (mo) to find the bestfocussing position.

In order to determine the pH value of the blood, from which a Ramansignal has been detected by the detection system (dsy), a signalprocessing system (sps) is provided in the optical detection system(ods). Therein at least one signal portion is extracted from the wholeRaman signal, which shows a significant pH sensitivity allowingdetermination of the pH value therefrom. From this at least one signalportion at least one predetermined band parameter is determined, such asband position, band intensity, band polarisation ratio, band widthand/or band shape or a combination of these. Which band parameter isactually determined depends on the fact, for which parameter arelationship between the pH value to be determined and band parameter isknown for the extracted signal portion of the Raman signal.

For instance, it has been found that the sensitive Raman bands in theheme group are 1378 cm⁻¹, 1506 cm⁻¹ and 1638 cm⁻¹ related to thevibrational band μ_(Fe-His) of the central Fe atom connected to theHistidin group of the heme protein hemoglobin. The influence of achanged pH to the position of porphyrin bands has also been measured inthe past showing a pH dependence of the wavenumber of ferrous alkylatedcytochrome c (Parker, Biological applications of IR and Raman, Chapter6, Porphyrins and Hemoproteins, page 276, 1982). It has been found thatalso human blood shows a similar pH dependence of the wavenumber at theabove mentioned band positions so that in a preferred embodiment theband position, or more particularly the wavenumber, is determined fromthe extracted signal portion of the Raman signal from which then by useof the given relationship the pH value can be determined. Such a pHdependence of the wavenumber and a band position around 1535 cm⁻¹ forblood is schematically shown in FIG. 2.

In order to directly determine the pH value of blood by use of adetected Raman signal besides measuring the band parameters of the pHsensitive heme vibrations other ways are possible as well, such asmeasuring the polarisation characteristics of the pH sensitive hemevibrations. Further, a multivariate statistical analysis of whole bloodspectra can be performed and train prediction models on blood samples ofknown pH can be used as described above. Further, the embodiment of ananalysis apparatus shown in FIG. 1 is only one example of a particularlayout. Of course, other embodiments of an analysis apparatus, forinstance having other embodiments of monitoring systems, e.g. adaptedfor monochromatic, bichromatic or multichromatic imaging, can be used aswell.

In summary, the following , steps or considerations can be taken toperform a non-invasive in vivo analysis of blood for determining the pHvalue:

-   -   1. recognize that certain heme band positions shift in        dependence on pH;    -   2. need to derive pH from band (shifted) positions Ω of heme        vibrations in blood;    -   3. therefore need to inverse the relationship of Ω=Ω(ph) to        pH=pH(Ω);    -   4. in blood of healthy people the pH range is small        (pH=7.25-7.45), pH<7.25 is danger for changed cell metabolism,        and medical action is needed;    -   5. find band positions of pH sensitive heme vibrations that show        a change in this range;    -   6. recognize that this change in band positions is in a        practically measurable wavenumber range (1-2 cm⁻¹);    -   7. need to measure band positions of heme with high enough        accuracy (0.02 to 0.1 cm⁻¹) to get relevant accuracy in pH        change;    -   8. do the experiment on in vivo blood: measure Raman of heme        vibrations of hemoglobin in in vivo blood;    -   9. the ideal way to do this is non-invasively: find blood        vessels in the skin and measure directly in the blood vessel;    -   10. check if there are interferences from other analytes (e.g.        lactate and lactate acidosis, oxygenation, etc), and if so        remove them;    -   11. optimize signals by finding the right excitation        wavelength(s) to do the measurement.

The invention allows a direct, quick, accurate and non-invasivedetermination of the pH value of blood without the need to separatelydetermine pCO₂ or bicarbonate.

1. A spectroscopic analysis apparatus for non-invasive in vivo analysisof blood comprising: an excitation system for emitting an excitationbeam to excite a target region containing a blood vessel, saidexcitation system configured to emit an excitation beam having apredetermined polarization orientation; a detection system for detectingscattered radiation from blood in the blood vessel in the tar et re iongenerated b the excitation beam to obtain a Raman signal; a signalprocessing system for extracting at least one predetermined pH sensitivesignal portion from said Raman signal and for determining adepolarization ratio from said at least one pH sensitive signal portionof said Raman signal and for determining the pH value of the blood inthe target region from said depolarization ratio by use of arelationship between pH value and depolarization ratio of vibrations ofone or more molecules at excitation wavelengths included in said atleast one pH sensitive signal portions; a monitoring system for imagingthe target region; and a focusing system for focusing the excitationsystem, the detection system and the monitoring system on the targetregion.
 2. The analysis apparatus as claimed in claim 1, wherein saiddetection system is adapted to detect scattered radiation from thetarget region containing essentially whole blood or red blood cells. 3.The analysis apparatus as claimed in claim 1, further including: a lightsource which provides a light beam for the monitoring system.
 4. Theanalysis apparatus as claimed in claim 3, further including: amicroscope objective which focuses the excitation beam and themonitoring system light source beam on the target region.
 5. Theanalysis apparatus as claimed in claim 1, further including: a filterfor suppressing directly reflected light from reaching the monitoringsystem and contributing to the imaging of the target region.
 6. Aspectroscopic analysis apparatus for non-invasive in vivo analysis ofblood comprising: an excitation system for emitting an excitation beamto excite a target region containing blood; a detection system fordetecting scattered radiation from the target region generated by theexcitation beam to obtain a Raman signal; a signal processing system forextracting at least one predetermined pH sensitive signal portion fromsaid Raman signal and for determining the pH value of the blood in thetarget region from said at least one pH sensitive signal portion by useof a relationship between pH value and one or more band parameters of pHsensitive vibration of at least one molecule or part of a moleculepresent in blood; a monitoring system for imaging the target region; afocusing system for focusing the excitation system, the detection systemand the monitoring system on the target region on a blood vessel; and anauto focus system which continually adjusts a confocal center of theexcitation beam.
 7. The analysis apparatus as claimed in claim 6,wherein said signal processing system is adapted for determining the pHvalue by use of a given relationship between pH value and band position,band intensity, band polarization ratio, band width and/or band shape.8. The analysis apparatus as claimed in claim 6, wherein said signalprocessing system is adapted for determining the pH value by use of arelationship between pH value and one or more band parameters of pHsensitive heme vibrations of hemoglobin.
 9. The analysis apparatus asclaimed in claim 8, wherein said detection system is adapted to detectonly one or more Raman signal portions of the heme vibrations ofhemoglobin.
 10. The analysis apparatus as claimed in claim 6, whereinsaid signal processing system is further adapted for checking if the atleast one pH sensitive signal portion includes interferences from otheranalytes and for removing such interferences from the at least one pHsensitive signal portion.
 11. The analysis apparatus as claimed in claim6, wherein said excitation system is adapted for emitting an excitationbeam at a wavelength which is optimized such that the at least one pHsensitive signal portion shows an optimum pH sensitivity.
 12. Theanalysis apparatus as claimed in claim 6, wherein said signal processingsystem is adapted for extracting at least one predetermined ph sensitivesignal portion from said Raman signal at a band position ofsubstantially 1378 cm⁻¹, 1506 cm⁻¹ or 1638 cm⁻¹.
 13. The analysisapparatus as claimed in claim 6, wherein the excitation system emits anexcitation beam having a predetermined polarization orientation andwherein the signal processing system determines a band parameter ofvibrations of one or more blood molecules in the target region from theRaman signal and determines the pH value of the blood in the targetregion from the determined band parameter.
 14. The analysis apparatus asclaimed in claim 13, wherein the band parameter includes depolarizationratio excitation profiles at a preselected excitation wavelength.
 15. Aspectroscopic analysis apparatus including: an optical detection systemincluding: an excitation s stem configured to emit an excitation beam toexcite blood in a blood vessel below a surface of a subject, a detectionsystem configured to detect scattered radiation from the blood in theblood vessel to generate a Raman signal, a signal processing systemconfigured to determine a pH value of the blood in the blood vessel fromthe Raman signal; an optical monitoring system including: an opticallight source configured to emit a monitoring beam that penetrates to theblood vessel, a camera configured to receive the monitoring beam fromthe blood vessel and form an image of the blood vessel; a microscopeobject which focuses the excitation and monitoring beams into thesubject, the microscope object hem controllable in accordance with theimage to focus at least the excitation beam on the blood vessel; and anauto focus mechanism connected with the camera and the microscope objectto adjust the focus of the microscope object to maintain a confocaldetection center of the excitation beam focused on the blood vessel whenthe subject moves.
 16. The analysis apparatus as claimed in claim 15,further including: a filter for suppressing directly reflected lightfrom reaching the monitoring system and contributing to the imaging ofthe blood vessel.
 17. The analysis system as claimed in claim 15,wherein the autofocus mechanism is further configured to track the bloodvessel through three spatial dimensions of motion.
 18. The analysisapparatus as claimed in claim 15, wherein the excitation system emits anexcitation beam having a predetermined polarization orientation andwherein the signal processing system determines a depolarization ratioof one or more molecules in the blood in the blood vessel at bloodexcitation wavelengths from the Raman signal and determines the pH valueof the blood in the blood vessel from the determined depolarizationratio.